NZ585774A

NZ585774A - Bivalent, bispecific antibodies

Info

Publication number: NZ585774A
Application number: NZ585774A
Authority: NZ
Inventors: Christian Klein; Wolfgang Schaefer
Original assignee: Hoffmann La Roche
Priority date: 2007-12-21
Filing date: 2008-12-16
Publication date: 2011-10-28
Also published as: HRP20140727T1; CN101903406A; AU2008340694B2; US8242247B2; AR118118A2; PE20091169A1; EP2225280A1; UA100874C2; CY1115275T1; MA31925B1; ZA201004300B; CO6280543A2; SI2225280T1; IL206108A0; CN101903406B; ECSP10010297A; JP2011505848A; MY160736A; IL206108A; KR20100087394A

Abstract

Disclosed is a bivalent, bispecific antibody, comprising: a) the light chain and heavy chain of an antibody specifically binding to a first antigen; and b) the light chain and heavy chain of an antibody specifically binding to a second antigen, wherein the constant domains CL and CH1 are replaced by each other. Also disclosed is a method for the preparation of the antibody.

Description

<div class="application article clearfix" id="description"> Received at IPONZ on 30.08.2011 Bivalent, bispecific antibodies The present invention relates to novel bivalent, bispecific antibodies, their manufacture and use. Background of the Invention Engineered proteins, such as bi- or multispecific antibodies capable of binding two 5 or more antigens are known in the art. Such multispecific binding proteins can be generated using cell fusion, chemical conjugation, or recombinant DNA techniques. A wide variety of recombinant bispecific antibody formats have been developed in the recent past, e.g. tetravalent bispecific antibodies by fusion of, e.g. an IgG 10 antibody format and single chain domains (see e.g. Coloma, M.J., et al, Nature Biotech 15 (1997) 159-163; WO 2001077342; and Morrison, S.L. , Nature Biotech 25 (2007) 1233-1234). Also several other new formats wherein the antibody core structure (IgA, IgD, IgE, IgG or IgM) is no longer retained such as dia-, tria- or tetrabodies, minibodies, 15 several single chain formats (scFv, Bis-scFv), which are capable of binding two or more antigens, have been developed(Holliger, P., et al, Nature Biotech 23 (2005) 1126-1136; Fischer, N., Leger O., Pathobiology 74 (2007) 3-14; Shen, J., et al, Journal of Immunological Methods 318 (2007) 65-74; Wu, C., et al, Nature Biotech 25 (2007) 1290-1297). 20 All such formats use linkers either to fuse the antibody core (IgA, IgD, IgE, IgG or IgM) to a further binding protein (e.g.. scFv) or to fuse e.g. two Fab fragments or scFv.(Fischer N., Leger O., Pathobiology 74 (2007) 3-14). While it is obvious that linkers have advantages for the engineering of bispecific antibodies, they may also cause problems in therapeutic settings. Indeed, these foreign peptides might elicit 25 an immune response against the linker itself or the junction between the protein and the linker. Further more, the flexible nature of these peptides makes them more prone to proteolytic cleavage, potentially leading to poor antibody stability, aggregation and increased immunogenicity. In addition one may want to retain effector functions, such as e.g. complement-dependent cytotoxicity (CDC) or 30 antibody dependent cellular cytotoxicity (ADCC), which are mediated through the Received at IPONZ on 30.08.2011 -2- Fc receptor binding , by maintaining a high degree of similarity to naturally occurring. Thus ideally, one should aim at developing bispecific antibodies that are very similar in general structure to naturally occurring antibodies (like IgA, IgD, IgE, 5 IgG or IgM) with minimal deviation from human sequences. In one approach bispecific antibodies that are very similar to natural antibodies have been produced using the quadroma technology (see Milstein, C. and A.C. Cuello, Nature, 305 (1983) 537-40) based on the somatic fusion of two different hybridoma cell lines expressing murine monoclonal antibodies with the desired 10 specificities of the bispecific antibody. Because of the random pairing of two different antibody heavy and light chains within the resulting hybrid-hybridoma (or quadroma) cell line, up to ten different antibody species are generated of which only one is the desired, functional bispecific antibody. Due to the of presence of mispaired byproducts, and significantly reduced production yields, means 15 sophisticated purification procedures are required (see e.g. Morrison, S.L. , Nature Biotech 25 (2007) 1233-1234). In general the same problem of mispaired byproducts remains if recombinant expression techniques are used. An approach to circumvent the problem of mispaired byproducts, which is known as 'knobs-into-holes', aims at forcing the pairing of two different antibody heavy 20 chains by introducing mutations into the CH3 domains to modify the contact interface. On one chain bulky amino acids were replaced by amino acids with short side chains to create a 'hole'. Conversely, amino acids with large side chains were introduced into the other CH3 domain, to create a 'knob'. By coexpressing these two heavy chains (and two identical light chains, which have to be appropriate for 25 both heavy chains), high yields of heterodimer formation ('knob-hole') versus homodimer formation ('hole-hole' or 'knob-knob') was observed (Ridgway, J.B., Presta LG, Carter P; and WO 1996027011). The percentage of heterodimer could be further increased by remodeling the interaction surfaces of the two CH3 domains using a phage display approach and the introduction of a disulfide bridge 30 to stabilize the heterodimers (Merchant, A.M., et al, Nature Biotech 16 (1998) 677-681; Atwell, S., Ridgway, J.B., Wells, J.A., Carter, P., J Mol Biol 270 (1997) 26-35). New approaches for the knobs-into-holes technology are described in e.g. in EP 1870459A1. Although this format appears very attractive, no data describing progression towards the clinic are currently available. One important constraint of 35 this strategy is that the light chains of the two parent antibodies have to be identical Received at IPONZ on 30.08.2011 - 3 - to prevent mispairing and formation of inactive molecules. Thus this technique is not appropriate for easily developing recombinant, bivalent, bispecific antibodies against two antigens starting from two antibodies against the first and the second antigen, as either the heavy chains of these antibodies an/or the identical light 5 chains have to be optimized. Simon T. et al, EMBO Journal, 9 (1990) 1051 -1056 relates to domain mutants of monospecific antibodies. Summary of the Invention The invention relates to a bivalent, bispecific antibody, comprising: 10 a) the light chain and heavy chain of an antibody specifically binding to a first antigen; and b) the light chain and heavy chain of an antibody specifically binding to a second antigen, wherein the constant domains CL and CHI are replaced by each other. 15 A further embodiment of the invention is a method for the preparation of an a bivalent, bispecific antibody according to the invention comprising the steps of a) transforming a host cell with 20 -vectors comprising nucleic acid molecules encoding the light chain and heavy chain of an antibody specifically binding to a first antigen -vectors comprising nucleic acid molecules encoding the light chain and heavy chain of an antibody specifically binding to a second antigen, wherein the constant domains CL and CHI are replaced by each other; 25 b) culturing the host cell under conditions that allow synthesis of said antibody molecule; and c) recovering said antibody molecule from said culture. A further embodiment of the invention is a host cell comprising Received at IPONZ on 30.08.2011 -4- - vectors comprising nucleic acid molecules encoding the light chain and heavy chain of an antibody specifically binding to a first antigen - vectors comprising nucleic acid molecules encoding the light chain and heavy chain of an antibody specifically binding to a second antigen, wherein the 5 constant domains CL and CHI are replaced by each other, wherein the host all is not a human cell within a human. A further embodiment of the invention is a composition, preferably a pharmaceutical or a diagnostic composition of the antibody according to the invention. 10 A further embodiment of the invention is a pharmaceutical composition comprising an antibody according to the invention and at least one pharmaceutically acceptable excipient. Also described herein is a method for the treatment of a patient in need of therapy, characterized by administering to the patient a therapeutically effective amount of 15 an antibody according to the invention. Detailed Description of the Invention The invention relates to a bivalent, bispecific antibody, comprising: a) the light chain and heavy chain of an antibody specifically binding to a first antigen; and 20 b) the light chain and heavy chain of an antibody specifically binding to a second antigen, wherein the constant domains CL and CHI are replaced by each other Therefore said bivalent, bispecific antibody, comprises: 25 a) a first light chain and a first heavy chain of an antibody specifically binding to a first antigen; and b) a second light chain and a second heavy chain of an antibody specifically binding to a second antigen, wherein the constant domains CL and CHI of the second light chain and the second heavy chain are replaced by each 30 other. Received at IPONZ on 30.08.2011 - 5 - Thus for said antibody specifically binding to a second antigen the following applies: within the light chain the constant light chain domain CL is replaced by the constant heavy chain 5 domain CHI of said antibody; and within the heavy chain the constant heavy chain domain CHI is replaced by the constant light chain domain CL of said antibody. The term "antibody" as used herein refers to whole, monoclonal antibodies. Such 10 whole antibodies consist of two pairs of a "light chain" (LC) and a "heavy chain" (HC) (such light chain (LC) /heavy chain pairs are abbreviated herein as LC/HC). The light chains and heavy chains of such antibodies are polypeptides consisting of several domains. In a whole antibody, each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or YH) and a heavy chain constant 15 region. The heavy chain constant region comprises the heavy chain constant domains CHI, CH2 and CH3 (antibody classes IgA, IgD, and IgG) and optionally the heavy chain constant domain CH4 (antibody classes IgE and IgM). Each light chain comprises a light chain variable domain YL and a light chain constant domain CL. The structure of one naturally occurring whole antibody, the IgG 20 antibody, is shown e.g. in Fig.l. The variable domains VH and VL can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each YH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: 25 FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 ((Janeway, C.A., Jr, et al, (2001). Immunobiology., 5th ed., Garland Publishing; and Woof, J., Burton, D., Nat Rev Immunol 4 (2004) 89-99). The two pairs of heavy chain and light chain (HC/LC) are capable of specifically binding to same antigen. Thus said whole antibody is a bivalent, monospecific antibody. Such "antibodies" include e.g. mouse antibodies, 30 human antibodies, chimeric antibodies, humanized antibodies and genetically engineered antibodies (variant or mutant antibodies) as long as their characteristic properties are retained. Especially preferred are human or humanized antibodies, especially as recombinant human or humanized antibodies. There are five types of mammalian antibody heavy chains denoted by the Greek 35 letters: a, 8, s, y, and (x (Janeway, C.A., Jr, et al, (2001). Immunobiology., 5th ed., Received at IPONZ on 30.08.2011 - 6- Garland Publishing). The type of heavy chain present defines the class of antibody; these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively (Rhoades RA, Pflanzer RG (2002). Human Physiology, 4th ed., Thomson Learning). Distinct heavy chains differ in size and composition; a and y contain 5 approximately 450 amino acids, while (i and s have approximately 550 amino acids. Each heavy chain has two regions, the constant region and the variable region. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotype. Heavy chains y, a and 8 have a constant region 10 composed of three constant domains CHI, CH2, and CH3 (in a line) , and a hinge region for added flexibility (Woof, J., Burton, D., Nat Rev Immunol 4 (2004) 89-99); heavy chains p. and s have a constant region composed of four constant domains CHI, CH2, CH3, and CH4 (Janeway, C.A., Jr. et al. (2001). Immunobiology., 5th ed., Garland Publishing). The variable region of the heavy 15 chain differs in antibodies produced by different B cells, but is the same for all antibodies produced by a single B cell or B cell clone. The variable region of each heavy chain is approximately 110 amino acids long and is composed of a single antibody domain. In mammals there are only two types of light chain, which are called lambda (A) 20 and kappa (k). A light chain has two successive domains: one constant domain CL and one variable domain VL. The approximate length of a light chain is 211 to 217 amino acids. Preferably the light chain is a kappa (k) light chain, and the constant domain CL is preferably C kappa (k). The terms "monoclonal antibody" or "monoclonal antibody composition" as used 25 herein refer to a preparation of antibody molecules of a single amino acid composition. The "antibodies" according to the invention can be of any class (e.g. IgA, IgD, IgE, IgG, and IgM, preferably IgG or IgE), or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2, preferably IgGl), whereby both antibodies, from which the 30 bivalent bispecific antibody according to the invention is derived, have an Fc part of the same subclass( e.g. IgGl, IgG4 and the like, preferably IgGl), preferably of the same allotype (e.g. Caucasian). Received at IPONZ on 30.08.2011 A "Fc part of an antibody" is a term well known to the skilled artisan and defined on the basis of papain cleavage of antibodies. The antibodies according to the invention contain as Fc part, preferably a Fc part derived from human origin and preferably all other parts of the human constant regions. The Fc part of an antibody 5 is directly involved in complement activation, Clq binding, C3 activation and Fc receptor binding. While the influence of an antibody on the complement system is dependent on certain conditions, binding to Clq is caused by defined binding sites in the Fc part. Such binding sites are known in the state of the art and described e.g. by Lukas, T.J., et al., J. Immunol. 127 (1981) 2555-2560; Brunhouse, R., and 10 Cebra, J.J., Mol. Immunol. 16 (1979) 907-917; Burton, D.R., et al., Nature 288 (1980) 338-344; Thommesen, J.E., et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie, E.E., et al., J. Immunol. 164 (2000) 4178-4184; Hezareh, M., et al., J. Virol. 75 (2001) 12161-12168; Morgan, A., etal., Immunology 86 (1995) 319-324; and EP0 307 434. Such binding sites are e.g. L234, L235, D270, N297, E318, 15 K320, K322, P331 and P329 (numbering according to EU index of Kabat, see below). Antibodies of subclass IgGl, IgG2 and IgG3 usually show complement activation, Clq binding and C3 activation, whereas IgG4 do not activate the complement system, do not bind Clq and do not activate C3. Preferably the Fc part is a human Fc part. 20 The term "chimeric antibody" refers to an antibody comprising a variable region, i.e., binding region, from one source or species and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. Chimeric antibodies comprising a murine variable region and a human constant region are preferred. Other preferred forms of "chimeric 25 antibodies" encompassed by the present invention are those in which the constant region has been modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to Clq binding and/or Fc receptor (FcR) binding. Such chimeric antibodies are also referred to as "class-switched antibodies.". Chimeric antibodies are the product of expressed 30 immunoglobulin genes comprising DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques are well known in the art. See, e.g., Morrison, S.L., et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; 35 US 5,202,238 and US 5,204,244. Received at IPONZ on 30.08.2011 - 8 - The term "humanized antibody" refers to antibodies in which the framework or "complementarity determining regions" (CDR) have been modified to comprise the CDR of an immunoglobulin of different specificity as compared to that of the parent immunoglobulin. In a preferred embodiment, a murine CDR is grafted into 5 the framework region of a human antibody to prepare the "humanized antibody." See, e.g., Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M.S., et al., Nature 314 (1985) 268-270. Particularly preferred CDRs correspond to those representing sequences recognizing the antigens noted above for chimeric antibodies. Other forms of "humanized antibodies" encompassed by the present 10 invention are those in which the constant region has been additionally modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to Clq binding and/or Fc receptor (FcR) binding. The term "human antibody", as used herein, is intended to include antibodies 15 having variable and constant regions derived from human germ line immunoglobulin sequences. Human antibodies are well-known in the state of the art (van Dijk, M.A., and van de Winkel, J.G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire or a 20 selection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258; Bruggemann, 25 M., et al., Year Immunol. 7 (1993) 33-40). Human antibodies can also be produced in phage display libraries (Hoogenboom, H.R., and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J.D., et al., J. Mol. Biol. 222 (1991) 581-597). The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer 30 Therapy, Alan R. Liss, p. 77 (1985); and Boerner, P., et al., J. Immunol. 147 (1991) 86-95). As already mentioned for chimeric and humanized antibodies according to the invention the term "human antibody" as used herein also comprises such antibodies which are modified in the constant region to generate the properties according to the invention, especially in regard to Clq binding and/or FcR binding, 35 e.g. by "class switching" i.e. change or mutation of Fc parts (e.g. from IgGl to IgG4 and/or IgGl/IgG4 mutation.) Received at IPONZ on 30.08.2011 - 9- The term "recombinant human antibody", as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell such as a NSO or CHO cell or from an animal (e.g. a mouse) that is transgenic for human immunoglobulin genes 5 or antibodies expressed using a recombinant expression vector transfected into a host cell. Such recombinant human antibodies have variable and constant regions in a rearranged form. The recombinant human antibodies according to the invention have been subjected to in vivo somatic hypermutation. Thus, the amino acid sequences of the YH and VL regions of the recombinant antibodies are sequences 10 that, while derived from and related to human germ line VH and VL sequences, may not naturally exist within the human antibody germ line repertoire in vivo. The "variable domain" (variable domain of a light chain (VL), variable region of a heavy chain (VH)) as used herein denotes each of the pair of light and heavy chains which is involved directly in binding the antibody to the antigen. The domains of variable human light and heavy chains have the same general structure and each domain comprises four framework (FR) regions whose sequences are widely conserved, connected by three "hypervariable regions" (or complementarity determining regions, CDRs). The framework regions adopt a P-sheet conformation and the CDRs may form loops connecting the P-sheet structure. The CDRs in each chain are held in their three-dimensional structure by the framework regions and form together with the CDRs from the other chain the antigen binding site. The antibody heavy and light chain CDR3 regions play a particularly important role in the binding specificity/affinity of the antibodies according to the invention and therefore provide a further object of the invention. 25 The terms "hypervariable region" or "antigen-binding portion of an antibody" when used herein refer to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region comprises amino acid residues from the "complementarity determining regions" or "CDRs". "Framework" or "FR" regions are those variable domain regions other than the hypervariable region 30 residues as herein defined. Therefore, the light and heavy chains of an antibody comprise from N- to C-terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDRs on each chain are separated by such framework amino acids. Especially, CDR3 of the heavy chain is the region which contributes most to antigen binding. CDR and FR regions are determined according to the standard 15 20 Received at IPONZ on 30.08.2011 -10- definition of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). The "constant domains" of the heavy chain and of the light chain are not involved directly in binding of an antibody to an antigen, but exhibit various effector 5 functions. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins are divided into the classes: The term "bivalent, bispecific antibody" as used herein refers to an antibody as described above in which each of the two pairs of heavy chain and light chain (HC/LC) is specifically binding to a different antigen, i.e. the first heavy and the 10 first light chain (originating from an antibody against a first antigen) are specifically binding together to a first antigen, and , the second heavy and the second light chain (originating from an antibody against a second antigen ) are specifically binding together to a second antigen (as depicted in Fig. 2); such bivalent, bispecific antibodies are capable of specifically binding to two different 15 antigens at the same time, and not to more than two antigens, in contrary to, on the one hand a monospecific antibody capable of binding only to one antigen, and on the other hand e.g. a tetravalent, tetraspecific antibody which can bind to four antigen molecules at the same time. According to the invention, the ratio of a desired bivalent, bispecific antibody 20 compared to undesired side products can be improved by the replacement of certain domains in only one pair of heavy chain and light chain (HC/LC). While the first of the two HC/LC pairs originates from an antibody specifically binding to a first antigen and is left essentially unchanged, the second of the two HC/LC pairs originates from an antibody specifically binding to a second antigen , and is altered 25 by the following replacement: light chain: replacement of the constant light chain domain CL by the constant heavy chain domain CHI of said antibody specifically binding to a second antigen, and heavy chain: replacement of the constant heavy chain domain CHI by the 30 constant light chain domain CL of said antibody specifically binding to a second antigen. Thus the resulting bivalent, bispecific antibodies are artificial antibodies which comprise Received at IPONZ on 30.08.2011 -11 - a) the light chain and heavy chain of an antibody specifically binding to a first antigen; and b) the light chain and heavy chain of an antibody specifically binding to a second antigen; 5 wherein said light chain (of an antibody specifically binding to a second antigen) contains a constant domain CHI instead of CL wherein said heavy chain(of an antibody specifically binding to a second antigen) a constant domain CL instead of CHI. 10 In an additional aspect of the invention such improved ratio of a desired bivalent, bispecific antibody compared to undesired side products can be further improved by one of the following two alternatives: A) First alternative (see Fig. 3): The CH3 domains of said bivalent, bispecific antibody according to the invention can be altered by the "knob-into-holes" technology which described with in detail with several examples in e.g. WO 96/027011, Ridgway, J.B., et al, Protein Eng 9 (1996) 617-621; and Merchant, A.M., et al, Nat Biotechnol 16 (1998) 677-681. In this method the interaction surfaces of the two CH3 domains are altered to increase the heterodimerisation of both heavy chains containing these two CH3 domains. Each of the two CH3 domains (of the two heavy chains) can be the "knob", while the other is the "hole". The introduction of a disulfide bridge stabilizes the heterodimers (Merchant, A..M., et al, Nature Biotech 16 (1998) 677-681; Atwell, S., Ridgway, J.B., Wells, J.A., Carter, P., J Mol Biol 270 (1997) 26-35) and increases the yield. Therefore in preferred embodiment the CH3 domains of a bivalent, bispecific antibody wherein the first CH3 domain and second CH3 domain each meet at an interface which comprises an original interface between the antibody CH3 domains are altered by the "knob-into-holes" technology including further stabilization by 30 introduction of a disulfide bridge in the CH3 domains (described in WO 96/027011, Ridgway, J.B., et al, Protein Eng 9 (1996) 617-621; Merchant. A.M, et al., Nature Biotech 16 (1998) 677-681; and Atwell, S., Ridgway, J.B., Wells, J.A., Carter P., J Mol Biol 270 (1997) 26-35) to promote the formation of the bivalent, bispecific antibody. 15 20 25 Received at IPONZ on 30.08.2011 - 12- Thus in one aspect of the invention said bivalent, bispecific antibody is characterized in that the CH3 domain of one heavy chain and the CH3 domain of the other heavy chain each meet at an interface which comprises an original interface between the 5 antibody CH3 domains; wherein said interface is altered to promote the formation of the bivalent, bispecific antibody, wherein the alteration is characterized in that: a) the CH3 domain of one heavy chain is altered, so that within the original interface the CH3 domain of one heavy chain that meets 10 the original interface of the CH3 domain of the other heavy chain within the bivalent, bispecific antibody, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the interface of the CH3 domain of one heavy chain which is positionable in a cavity within the interface of 15 the CH3 domain of the other heavy chain and b) the CH3 domain of the other heavy chain is altered, so that within the original interface of the second CH3 domain that meets the original interface of the first CH3 domain within the bivalent, bispecific antibody 20 an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the interface of the second CH3 domain within which a protuberance within the interface of the first CH3 domain is positionable. Preferably said amino acid residue having a larger side chain volume is selected 25 from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W). Preferably said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), valine (V). In one aspect of the invention both CH3 domains are further altered the 30 introduction of cysteine (C) as amino acid in the corresponding positions of each Received at IPONZ on 30.08.2011 - 13 - CH3 domain such that a disulfide bridge between both CH3 domains can be formed. In another preferred embodiment of the invention both CH3 domains are altered by the use of residues R409D; K370E (K409D) for knobs residues and D399K; 5 E357K for hole residues described eg. in EP 1870459A1. or B) Second alternative (see Figure 4): by the replacement of one constant heavy chain domain CH3 by a constant heavy 10 chain domain CHI; and the other constant heavy chain domain CH3is replaced by a constant light chain domain CL. The constant heavy chain domain CHI by which the heavy chain domain CH3 is replaced can be of any Ig class (e.g. IgA, IgD, IgE, IgG, and IgM), or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2). The constant light chain domain CL by which the heavy chain domain CH3 is 15 replaced can be of the lambda (A) or kappa (k) type, preferably the kappa (k) type. Thus one preferred embodiment of the invention is a bivalent, bispecific antibody, comprising: a) the light chain and heavy chain of an antibody specifically binding to a first 20 antigen; and b) the light chain and heavy chain of an antibody specifically binding to a second antigen, wherein the constant domains CL and CHI are replaced by each other, 25 and wherein optionally c) the CH3 domain of one heavy chain and the CH3 domain of the other heavy chain each meet at an interface which comprises an original interface between the antibody CH3 domains; 30 wherein said interface is altered to promote the formation of the bivalent, bispecific antibody, wherein the alteration is characterized in that: ca) the CH3 domain of one heavy chain is altered, Received at IPONZ on 30.08.2011 - 14- so that within the original interface the CH3 domain of one heavy chain that meets the original interface of the CH3 domain of the other heavy chain within the bivalent, bispecific antibody, an amino acid residue is replaced with an amino acid residue having a 5 larger side chain volume, thereby generating a protuberance within the interface of the CH3 domain of one heavy chain which is positionable in a cavity within the interface of the CH3 domain of the other heavy chain and cb) the CH3 domain of the other heavy chain is altered, 10 so that within the original interface of the second CH3 domain that meets the original interface of the first CH3 domain within the bivalent, bispecific antibody an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the interface 15 of the second CH3 domain within which a protuberance within the interface of the first CH3 domain is positionable; or d) one constant heavy chain domain CH3 is replaced by a constant heavy chain domain CHI; and the other constant heavy chain domain CH3 is 20 replaced by a constant light chain domain CL The terms "antigen" or "antigen molecule" as used herein are used interchangeable and refer to all molecules that can be specifically bound by an antibody. The bivalent, bispecific antibody is specifically binding to a first antigen and a second 25 distinct antigen. The term "antigens" as used herein include e.g. proteins, different epitopes on proteins (as different antigens within the meaning of the invention), and polysaccharides. This mainly includes parts (coats, capsules, cell walls, flagella, fimbrae, and toxins) of bacteria, viruses, and other microorganisms. Lipids and nucleic acids are antigenic only when combined with proteins and 30 polysaccharides. Non-microbial exogenous (non-self) antigens can include pollen, egg white, and proteins from transplanted tissues and organs or on the surface of transfused blood cells. Preferably the antigen is selected from the group consisting Received at IPONZ on 30.08.2011 - 15 - of cytokines, cell surface proteins, enzymes and receptors cytokines, cell surface proteins, enzymes and receptors. Tumor antigens are those antigens that are presented by MHC I or MHC II molecules on the surface of tumor cells. These antigens can sometimes be 5 presented by tumor cells and never by the normal ones. In this case, they are called tumor-specific antigens (TSAs) and typically result from a tumor specific mutation. More common are antigens that are presented by tumor cells and normal cells, and they are called tumor-associated antigens (TAAs). Cytotoxic T lymphocytes that recognized these antigens may be able to destroy the tumor cells before they 10 proliferate or metastasize. Tumor antigens can also be on the surface of the tumor in the form of, for example, a mutated receptor, in which case they will be recognized by B cells. In one preferred embodiment at least one of the two different antigens (first and second antigen), to which the bivalent, bispecific antibody specifically binds to, is a 15 tumor antigen. In another preferred embodiment both of the two different antigens (first and second antigen), to which the bivalent, bispecific antibody specifically binds to, are tumor antigens; in this case the first and second antigen can also be two different epitopes at the same tumor specific protein. 20 In another preferred embodiment one of the two different antigens (first and second antigen), to which the bivalent, bispecific antibody specifically binds to, is a tumor antigen and the other is an effector cell antigen, as e.g. a T-Cell receptor, CD3, CD 16 and the like. In another preferred embodiment one of the two different antigens (first and second 25 antigen), to which the bivalent, bispecific antibody specifically binds to, is a tumor antigen and the other is an anti-cancer substance such as a toxin or a kinase inhibitor. As used herein, "specifically binding" or "binds specifically to" refers to an antibody specifically binding an antigen. Preferably the binding affinity of the 30 antibody specifically binding this antigen is of KD-value of 10"9 mol/1 or lower (e.g. 10"10 mol/1), preferably with a KD-value of 10"10 mol/1 or lower (e.g. 10"12 mol/1). The binding affinity is determined with a standard binding assay, such as surface plasmon resonance technique (Biacore®). Received at IPONZ on 30.08.2011 - 16- The term "epitope" includes any polypeptide determinant capable of specific binding to an antibody. In certain embodiments, epitope determinant include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. In certain embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. An further embodiment of the invention is a method for the preparation of a bivalent, bispecific antibody according to the invention comprising a) transforming a host cell with -vectors comprising nucleic acid molecules encoding the light chain and heavy chain of an antibody specifically binding to a first antigen -vectors comprising nucleic acid molecules encoding the light chain and heavy chain of an antibody specifically binding to a second antigen, wherein the constant domains CL and CHI are replaced by each other; b) culturing the host cell under conditions that allow synthesis of said antibody molecule; and c) recovering said antibody molecule from said culture. In general there are two vectors encoding the light chain and heavy chain of said antibody specifically binding to a first antigen, and further two vectors encoding the light chain and heavy chain of said antibody specifically binding to a second antigen. One of the two vectors is encoding the respective light chain and the other of the two vectors is encoding the respective heavy chain. However in an alternative method for the preparation of a bivalent, bispecific antibody according to the invention, only one first vector encoding the light chain and heavy chain of the antibody specifically binding to a first antigen and only one second vector encoding the light chain and heavy chain of the antibody specifically binding to a second antigen can be used for transforming the host cell. Received at IPONZ on 30.08.2011 - 17- The invention encompasses a method for the preparation of the antibodies comprising culturing the corresponding host cells under conditions that allow synthesis of said antibody molecules and recovering said antibodies from said culture, e.g. by expressing 5 -a first nucleic acid sequence encoding the light chain of an antibody specifically binding to a first antigen; -a second nucleic acid sequence encoding the heavy chain of said antibody specifically binding to a first antigen; -a third nucleic acid sequence encoding the light chain of an antibody specifically 10 binding to a second antigen, wherein the constant light chain domain CL is replaced by the constant heavy chain domain CHI; and -a fourth nucleic acid sequence encoding the heavy chain of said antibody specifically binding to a second antigen, wherein constant heavy chain domain CHI by the constant light chain domain CL. 15 A further embodiment of the invention is a host cell comprising - vectors comprising nucleic acid molecules encoding the light chain and heavy chain of an antibody specifically binding to a first antigen - vectors comprising nucleic acid molecules encoding the light chain and heavy chain of an antibody specifically binding to a second antigen, wherein the constant 20 domains CL and CHI are replaced by each other. A further embodiment of the invention is a host cell comprising a) a vector comprising a nucleic acid molecule encoding the light chain and a vector comprising a nucleic acid molecule encoding the heavy chain, of an antibody specifically binding to a first antigen 25 b) a vector comprising a nucleic acid molecule encoding the light chain and a vector comprising a nucleic acid molecule encoding the heavy chain, of an antibody specifically binding to a second antigen, wherein the constant domains CL and CHI are replaced by each other. Received at IPONZ on 30.08.2011 - 18 - A further embodiment of the invention is a composition, preferably a pharmaceutical or a diagnostic composition of the bivalent, bispecific antibody according to the invention. A further embodiment of the invention is a pharmaceutical composition comprising 5 a bivalent, bispecific antibody according to the invention and at least one pharmaceutically acceptable excipient. Also described herein is a method for the treatment of a patient in need of therapy, characterized by administering to the patient a therapeutically effective amount of a bivalent, bispecific antibody according to the invention. 10 The term "nucleic acid or nucleic acid molecule", as used herein, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA. As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words 15 "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. 20 Where distinct designations are intended, it will be clear from the context. The term "transformation" as used herein refers to process of transfer of a vectors/nucleic acid into a host cell. If cells without formidable cell wall barriers are used as host cells, transfection is carried out e.g. by the calcium phosphate precipitation method as described by Graham and Van der Eh, Virology 52 (1978) 25 546ff. However, other methods for introducing DNA into cells such as by nuclear injection or by protoplast fusion may also be used. If prokaryotic cells or cells which contain substantial cell wall constructions are used, e.g. one method of transfection is calcium treatment using calcium chloride as described by Cohen, F. N, et al, PNAS. 69 (1972) 711 Off. 30 Recombinant production of antibodies using transformation is well-known in the state of the art and described, for example, in the review articles of Makrides, S.C., Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, R.J., Mol. Biotechnol. 16 (2000) 151-161; Werner, Received at IPONZ on 30.08.2011 - 19- R.G., et al., Arzneimittelforschung 48 (1998) 870-880 as well as in US 6,331,415 and US 4,816,567. As used herein, "expression" refers to the process by which a nucleic acid is transcribed into mRNA and/or to the process by which the transcribed mRNA (also 5 referred to as transcript) is subsequently being translated into peptides, polypeptides, or proteins. The transcripts and the encoded polypeptides are collectively referred to as gene product. If the polynucleotide is derived from genomic DNA, expression in a eukaryotic cell may include splicing of the mRNA. A "vector" is a nucleic acid molecule, in particular self-replicating, which transfers 10 an inserted nucleic acid molecule into and/or between host cells. The term includes vectors that function primarily for insertion of DNA or RNA into a cell (e.g., chromosomal integration), replication of vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for transcription and/or translation of the DNA or RNA. Also included are vectors that provide more 15 than one of the functions as described. An "expression vector" is a polynucleotide which, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide. An "expression system" usually refers to a suitable host cell comprised of an expression vector that can function to yield a desired expression product. 20 The bivalent, bispecific antibodies according to the invention are preferably produced by recombinant means. Such methods are widely known in the state of the art and comprise protein expression in prokaryotic and eukaryotic cells with subsequent isolation of the antibody polypeptide and usually purification to a pharmaceutically acceptable purity. For the protein expression, nucleic acids 25 encoding light and heavy chains or fragments thereof are inserted into expression vectors by standard methods. Expression is performed in appropriate prokaryotic or eukaryotic host cells like CHO cells, NSO cells, SP2/0 cells, HEK293 cells, COS cells, yeast, or E.coli cells, and the antibody is recovered from the cells (supernatant or cells after lysis).The bivalent, bispecific antibodies may be present 30 in whole cells, in a cell lysate, or in a partially purified or substantially pure form. Purification is performed in order to eliminate other cellular components or other contaminants, e.g. other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, column chromatography and others well known Received at IPONZ on 30.08.2011 -20- in the art. See Ausubel, F., et al., ed., Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987). Expression in NSO cells is described by, e.g., Barnes, L.M., et al., Cytotechnology 32 (2000) 109-123; and Barnes, L.M., et al., Biotech. Bioeng. 73 (2001) 261-270. 5 Transient expression is described by, e.g., Durocher, Y., et al., Nucl. Acids. Res. 30 (2002) E9. Cloning of variable domains is described by Orlandi, R., et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; and Norderhaug, L., et al., J. Immunol. Methods 204 (1997) 77-87. A preferred transient expression system (HEK 293) is described by 10 Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30 (1999) 71-83 and by Schlaeger, E.-J., in J. Immunol. Methods 194 (1996) 191-199. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, enhancers and polyadenylation signals. 15 Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the 20 sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If 25 such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. The bivalent, bispecific antibodies are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, 30 dialysis, or affinity chromatography. DNA and RNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures. The hybridoma cells can serve as a source of such DNA and RNA. Once isolated, the DNA may be inserted into expression vectors, which are then transfected into host cells such as HEK 293 cells, CHO cells, or myeloma cells that do not otherwise Received at IPONZ on 30.08.2011 - 21 - produce immunoglobulin protein, to obtain the synthesis of recombinant monoclonal antibodies in the host cells. Amino acid sequence variants (or mutants) of the bivalent, bispecific antibody are prepared by introducing appropriate nucleotide changes into the antibody DNA, or 5 by nucleotide synthesis. Such modifications can be performed, however, only in a very limited range, e.g. as described above. For example, the modifications do not alter the above mentioned antibody characteristics such as the IgG isotype and antigen binding, but may improve the yield of the recombinant production, protein stability or facilitate the purification. 10 The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention. Sequence Listing 15 SEQ ID NO: 1 amino acid sequence of wild type <IGF-1R> antibody heavy chain SEQ ID NO: 2 amino acid sequence of wild type <IGF-1R> antibody light chain SEQ ID NO: 3 amino acid sequence of the heavy chain** (HC**) of <IGF- 20 1R> CL-CH1 exchange antibody, wherein the heavy chain domain CHI is replaced by the light chain domain CL. SEQ ID NO: 4 amino acid sequence of the light chain** (LC**) of <IGF-1R> CL-CH1 exchange antibody, wherein the light chain domain CL is replaced by the heavy chain domain CHI. 25 SEQ II) NO: 5 amino acid sequence of IGF-1R ectodomain His-Streptavidin binding peptide-tag (IGF-IR-His-SBP ECD) SEQ ID NO: 6 amino acid sequence of wild type ANGPT2 <ANGPT2> antibody heavy chain SEQ ID NO: 7 amino acid sequence of wild type ANGPT2 <ANGPT2> 30 antibody light chain SEQ ID NO: 8 amino acid sequence of CH3 domain (Knobs) with a T366W exchange for use in the knobs-into-holes technology Received at IPONZ on 30.08.2011 -22- SEQ ID NO: 9 SEQ ID NO: 10 amino acid sequence CH3 domain (Hole) with a T366S, L368A, Y407V exchange for use in the knobs-into-holes technology amino acid sequence of IGF-1R ectodomain His-Streptavidin binding peptide-tag (IGF-IR-His-SBP ECD) Description of the Figures Figure 1 Schematic figure of IgG, a naturally occurring whole antibody specific for one antigen with two pairs of heavy and light chain 10 which comprise variable and constant domains in a typical order. Figure 2 Schematic figure of a bivalent, bispecific antibody, comprising: a) the light chain and heavy chain of an antibody specifically binding to a first antigen; and b) the light chain and heavy chain of an antibody specifically binding to a second antigen, wherein 15 the constant domains CL and CHI are replaced by each other. Figure 3 Schematic figure of a bivalent, bispecific antibody, comprising: a) the light chain and heavy chain of an antibody specifically binding to a first antigen; and b) the light chain and heavy chain of an antibody specifically binding to a second antigen, wherein 20 the constant domains CL and CHI are replaced by each other, and wherein the CH3 domains of both heavy chains are altered by the knobs-into-holes technology . Figure 4 Schematic figure of a bivalent, bispecific antibody, comprising: a) the light chain and heavy chain of an antibody specifically 25 binding to a first antigen; and b) the light chain and heavy chain of an antibody specifically binding to a second antigen, wherein the constant domains CL and CHI are replaced by each other, and wherein one of the constant heavy chain domains CH3 of both heavy chains is replaced by a constant heavy chain domain 30 CHI, and the other constant heavy chain domain CH3 is replaced by a constant light chain domain CL. Figure 5 Protein sequence scheme of the heavy chain** <IGF-1R> HC** of the <IGF-1R> CL-CH1 exchange antibody (with a kappa constant light chain domain CL ) 35 Figure 6 Protein sequence scheme of the light chain** <IGF-1R> LC** of the <IGF-1R> CL-CH1 exchange antibody Received at IPONZ on 30.08.2011 - 23 - 10 Figure 11 Figure 12 15 20 25 30 Figure 7 Plasmid map of heavy chain** <IGF-1R> HC** expression vector PUC-HC**-IGF-1R Figure 8 Plasmid map of light chain** <IGF-1R> LC** expression vector pUC-LC**-IGF-lR Figure 9 Plasmid map of the 47QO-Hyg-OriP expression vector Figure 10 Assay principle of cellular FACS IGF-1R-ANGPT2 bridging assay on 124 IGF-IR expressing cells to detect the presence of functional bispecific <ANGPT2-IGF-1R> CL-CH1 exchange antibody Scheme IGF-IR ECD Biacore SDS-PAGE and size exclusion chromatography of purified monospecific, bivalent <IGF-1R> CL-CH1 exchange antibody (IgGl*) with HC* and LC* isolated from cell culture supernatants after transient transfection of HEK293-F cells. Figure 13 Binding of monospecific <IGF-1R> CL-CH1 exchange antibody and wildtype <IGF-1R> antibody to the IGF-IR ECD in an ELISA-based binding assay. Figure 14 SDS-PAGE of and size exclusion chromatography <ANGPT2- IGF-1R> CL-CH1 exchange antibody mix purified from cell culture supernatants from transiently transfected HEK293-F cells. Figure 15 Results for Samples A to F of cellular FACS IGF-IR-ANGPT2 bridging assay on 124 IGF-IR expressing cells to detect the presence of functional bispecific <ANGPT2-IGF-1R> CL-CH1 exchange antibody in purified antibody mix: Purified proteins Samples A to F: A = 124 untreated B = 124 + 2 ng/mL hANGPT2 + hlgG Isotype C = 124 + 2 |jg/mL hANGPT2 + Mix from co-expression of <IGF-1R> CL-CH1 exchange antibody and <ANGPT2> wildtype antibody comprising bispecific <ANGPT2-IGF-1R> CL-CH1 exchange antibody D: not present E = 124 + 2 (jg/mL hANGPT2 + <ANGPT2> wildtype antibody F = 124 + 2 |ig/mL hANGPT2 + <IGF-1R> wildtype antibody 35 Received at IPONZ on 30.08.2011 -24- Examples Materials & general methods General information regarding the nucleotide sequences of human immunoglobulins light and heavy chains is given in: Kabat, E.A., et al., Sequences 5 of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). Amino acids of antibody chains are numbered and referred to according to EU numbering (Edelman, G.M., et al., Proc. Natl. Acad. Sci. USA 63 (1969) 78-85; Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of 10 Health, Bethesda, MD, (1991)). Recombinant DNA techniques Standard methods were used to manipulate DNA as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. The molecular biological reagents were 15 used according to the manufacturer's instructions. Gene synthesis Desired gene segments were prepared from oligonucleotides made by chemical synthesis. The 600 - 1800 bp long gene segments, which are flanked by singular restriction endonuclease cleavage sites, were assembled by annealing and ligation 20 of oligonucleotides including PCR amplification and subsequently cloned via the indicated restriction sites e.g. Kpnl/ SacI or AscI/PacI into a pPCRScript (Stratagene) based pGA4 cloning vector. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing. Gene synthesis fragments were ordered according to given specifications at Geneart (Regensburg, Germany). 25 DNA sequence determination DNA sequences were determined by double strand sequencing performed at MediGenomix GmbH (Martinsried, Germany) or Sequiserve GmbH (Vaterstetten, Germany). Received at IPONZ on 30.08.2011 - 25 - DNA and protein sequence analysis and sequence data management The GCG's (Genetics Computer Group, Madison, Wisconsin) software package version 10.2 and Infomax's Vector NT1 Advance suite version 8.0 was used for sequence creation, mapping, analysis, annotation and illustration. 5 Expression vectors For the expression of the described antibodies variants of expression plasmids for transient expression (e.g. in HEK293 EBNA or HEK293-F) cells based either on a cDNA organization with a CMV-Intron A promoter or on a genomic organization 10 with a CMV promoter were applied. Beside the antibody expression cassette the vectors contained: - an origin of replication which allows replication of this plasmid in E. coli, and - a 13-lactamase gene which confers ampicillin resistance in E. coli. The transcription unit of the antibody gene is composed of the following elements: 15 - unique restriction site(s) at the 5' end - the immediate early enhancer and promoter from the human cytomegalovirus, - followed by the Intron A sequence in the case of the cDNA organization, - a 5'-untranslated region of a human antibody gene, - a immunoglobulin heavy chain signal sequence, 20 - the human antibody chain (wildtype or with domain echange) either as cDNA or as genomic organization with an the immunoglobulin exon-intron organization - a 3' untranslated region with a polyadenylation signal sequence, and - unique restriction site(s) at the 3' end. Received at IPONZ on 30.08.2011 -26- The fusion genes comprising the described antibody chains as decribed below were generated by PCR and/or gene synthesis and assembled with known recombinant methods and techniques by connection of the according nucleic acid segments e.g. using unique restriction sites in the respective vectors. The subcloned nucleic acid 5 sequences were verified by DNA sequencing. For transient transfections larger quantities of the plasmids were prepared by plasmid preparation from transformed E. coli cultures (Nucleobond AX, Macherey-Nagel). Cell culture techniques Standard cell culture techniques were used as described in Current Protocols in 10 Cell Biology (2000), Bonifacino, J.S., Dasso, M., Harford, J.B., Lippincott-Schwartz, J. and Yamada, K.M. (eds.), John Wiley & Sons, Inc. Bispecific antibodies were expressed by transient co-transfection of the respective expression plasmids in adherently growing HEK293-EBNA or in HEK29-F cells growing in suspension as described below. 15 Transient transfections in HEK293-EBNA system Bispecific antibodies were expressed by transient co-transfection of the respective expression plasmids (e.g. encoding the heavy and modified heavy chain, as well as the corresponding light and modified light chain) in adherently growing HEK293-EBNA cells (human embryonic kidney cell line 293 expressing Epstein-Barr-Virus 20 nuclear antigen; American type culture collection deposit number ATCC # CRL-10852, Lot. 959 218) cultivated in DMEM (Dulbecco's modified Eagle's medium, Gibco) supplemented with 10% Ultra Low IgG FCS (fetal calf serum, Gibco), 2 mM L-Glutamine (Gibco), and 250 |ig/ml Geneticin (Gibco). For transfection FuGENE™ 6 Transfection Reagent (Roche Molecular Biochemicals) was used in a 25 ratio of FuGENE™ reagent (|il) to DNA (|ig) of 4:1 (ranging from 3:1 to 6:1). Proteins were expressed from the respective plasmids using a molar ratio of (modified and wildtype) light chain and heavy chain encoding plasmids of 1:1 (equimolar) ranging from 1:2 to 2:1, respectively. Cells were feeded at day 3 with L-Glutamine ad 4 mM, Glucose [Sigma] and NAA [Gibco], Bispecific antibody 30 containing cell culture supernatants were harvested from day 5 to 11 after transfection by centrifugation and stored at -20°C. General information regarding Received at IPONZ on 30.08.2011 -27- the recombinant expression of human immunoglobulins in e.g. HEK293 cells is given in: Meissner, P. etal., Biotechnol. Bioeng. 75 (2001) 197-203. Transient transfections in HEK293-F system Bispecific antibodies were generated by transient transfection of the respective 5 plasmids (e.g. encoding the heavy and modified heavy chain, as well as the corresponding light and modified light chain) using the HEK293-F system (Invitrogen) according to the manufacturer's instruction. Briefly, HEK293-F cells (Invitrogen) growing in suspension either in a shake flask or in a stirred fermenter in serumfree FreeStyle 293 expression medium (Invitrogen) were transfected with a 10 mix of the four expression plasmids and 293fectin or fectin (Invitrogen). For 2 L shake flask (Corning) HEK293-F cells were seeded at a density of 1.0E*6 cells/mL in 600 mL and incubated at 120 rpm, 8% C02. The day after the cells were transfected at a cell density of ca. 1.5E*6 cells/mL with ca. 42 mL mix of A) 20 mL Opti-MEM (Invitrogen) with 600 |ig total plasmid DNA (1 |ig/mL) encoding 15 the heavy or modified heavy chain, respectively and the corresponding light chain in an equimolar ratio and B) 20 ml Opti-MEM +1.2 mL 293 fectin or fectin (2 pl/mL). According to the glucose consumption glucose solution was added during the course of the fermentation. The supernatant containing the secreted antibody was harvested after 5-10 days and antibodies were either directly purified from the 20 supernatant or the supernatant was frozen and stored. Protein determination The protein concentration of purified antibodies and derivatives was determined by determining the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence according to Pace et 25 al., Protein Science, 1995, 4, 2411-1423. Antibody concentration determination in supernatants The concentration of antibodies and derivatives in cell culture supernatants was estimated by immunoprecipitation with Protein A Agarose-beads (Roche). 60 |iL Protein A Agarose beads are washed three times in TBS-NP40 (50 mM Tris, pH 30 7.5, 150 mM NaCl, 1% Nonidet-P40). Subsequently, 1 -15 mL cell culture Received at IPONZ on 30.08.2011 - 28 - supernatant were applied to the Protein A Agarose beads pre-equilibrated in TBS-NP40. After incubation for at 1 h at room temperature the beads were washed on an Ultrafree-MC-filter column (Amicon] once with 0.5 mL TBS-NP40, twice with 0.5 mL 2x phosphate buffered saline (2xPBS, Roche) and briefly four times with 0.5 5 mL 100 mM Na-citrate pH 5,0. Bound antibody was eluted by addition of 35 |il NuPAGE® LDS Sample Buffer (Invitrogen). Half of the sample was combined with NuPAGE® Sample Reducing Agent or left unreduced, respectively, and heated for 10 min at 70°C. Consequently, 5-30 (jl were applied to an 4-12% NuPAGE® Bis-Tris SDS-PAGE (Invitrogen) (with MOPS buffer for non-reduced 10 SDS-PAGE and MES buffer with NuPAGE® Antioxidant running buffer additive (Invitrogen) for reduced SDS-PAGE) and stained with Coomassie Blue. The concentration of antibodies and derivatives in cell culture supernatants was quantitatively measured by affinity HPLC chromatography. Briefly, cell culture supernatants containing antibodies and derivatives that bind to Protein A were 15 applied to an Applied Biosystems Poros A/20 column in 200 mM KH2P04, 100 mM sodium citrate, pH 7.4 and eluted from the matrix with 200 mM NaCl, 100 mM citric acid, pH 2,5 on an Agilent HPLC 1100 system. The eluted protein was quantified by UV absorbance and integration of peak areas. A purified standard IgGl antibody served as a standard. Alternatively, the concentration of antibodies and derivatives in cell culture supernatants was measured by Sandwich-IgG-ELISA. Briefly, StreptaWell High Bind Strepatavidin A-96 well microtiter plates (Roche) were coated with 100 H-L/well biotinylated anti-human IgG capture molecule F(ab')2<h-Fcy> BI (Dianova) at 0.1 |jg/mL for 1 h at room temperature or alternatively over night at 4°C and subsequently washed three times with 200 |iL/well PBS, 0.05% Tween (PBST, Sigma). 100 (jL/well of a dilution series in PBS (Sigma) of the respective antibody containing cell culture supernatants was added to the wells and incubated for 1 -2 h on a microtiterplate shaker at room temperature. The wells were washed three times with 200 |iL/well PBST and bound antibody was detected with 100 |il F(ab')2<hFcy>POD (Dianova) at 0.1 |ig/mL as detection antibody for 1-2 h on a microtiterplate shaker at room temperature. Unbound detection antibody was washed away three times with 200 |iL/well PBST and the bound detection antibody was detected by addition of 100 |iL ABTS/well. Determination of absorbance was 20 25 30 Received at IPONZ on 30.08.2011 -29- performed on a Tecan Fluor Spectrometer at a measurement wavelength of 405 nm (reference wavelength 492 nm). Protein purification Proteins were purified from filtered cell culture supernatants referring to standard 5 protocols. In brief, antibodies were applied to a Protein A Sepharose column (GE healthcare) and washed with PBS. Elution of antibodies was achieved at pH 2.8 followed by immediate neutralization of the sample. Aggregated protein was separated from monomeric antibodies by size exclusion chromatography (Superdex 200, GE Healthcare) in PBS or in 20 mM Histidine, 150 mM NaCl pH 6.0. 10 Monomeric antibody fractions were pooled, concentrated if required using e.g. a MILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrator, frozen and stored at -20°C or -80°C. Part of the samples were provided for subsequent protein analytics and analytical characterization e.g. by SDS-PAGE, size exclusion chromatography or mass spectrometry. 15 SDS-PAGE The NuPAGE® Pre-Cast gel system (Invitrogen) was used according to the manufacturer's instruction. In particular, 10% or 4-12% NuPAGE® Novex® Bis-TRIS Pre-Cast gels (pH 6.4) and a NuPAGE® MES (reduced gels, with NuPAGE® Antioxidant running buffer additive) or MOPS (non-reduced gels) 20 running buffer was used. Analytical size exclusion chromatography Size exclusion chromatography for the determination of the aggregation and oligomeric state of antibodies was performed by HPLC chromatography. Briefly, Protein A purified antibodies were applied to a Tosoh TSKgel G3000SW column 25 in 300 mM NaCl, 50 mM KH2P04/K2HP04, pH 7.5 on an Agilent HPLC 1100 system or to a Superdex 200 column (GE Healthcare) in 2 x PBS on a Dionex HPLC-System. The eluted protein was quantified by UV absorbance and integration of peak areas. BioRad Gel Filtration Standard 151-1901 served as a standard. 30 Mass spectrometry Received at IPONZ on 30.08.2011 - 30- The total deglycosylated mass of crossover antibodies was determined and confirmed via electrospray ionization mass spectrometry (ESI-MS). Briefly, 100 (jg purified antibodies were deglycosylated with 50 mU N-Glycosidase F (PNGaseF, ProZyme) in 100 mM KH2P04/K2HP04, pH 7 at 37°C for 12-24 h at a protein 5 concentration of up to 2 mg/ml and subsequently desalted via HPLC on a Sephadex G25 column (GE Healthcare). The mass of the respective heavy and light chains was determined by ESI-MS after deglycosylation and reduction. In brief, 50 |ig antibody in 115 jj.1 were incubated with 60 |jl 1M TCEP and 50 jj.1 8 M Guanidine-hydrochloride subsequently desalted. The total mass and the mass of the reduced 10 heavy and light chains was determined via ESI-MS on a Q-Star Elite MS system equipped with a NanoMate source. IGF-IR ECD binding ELISA The binding properties of the generated antibodies were evaluated in an ELISA assay with the IGF-IR extracellular domain (ECD). For this sake the extracellular 15 domain of IGF-IR (residues 1-462) comprising the natural leader sequence and the Ll-cysteine rich-12 domains of the human IGF-IR ectodomain of the alpha chain (according to the McKern et al., 1997; Ward et al., 2001) fused to an N-terminal His-Streptavidin binding peptide-tag (His-SBP) was cloned into a pcDNA3 vector derivative and transiently expressed in HEK293F cells. The protein sequence of the 20 IGF-IR-His-SBP ECD is given in SEQ ID NO: 10. StreptaWell High Bind Strepatavidin A-96 well microtiter plates (Roche) were coated with 100 |iL/well cell culture supernatant containing soluble IGF-1R-ECD-SBP fusion protein over night at 4°C and washed three times with 200 (jL/well PBS, 0.05% Tween (PBST, Sigma). Subsequently, 100 |iL/well of a dilution series of the respective antibody 25 and as a reference wildtype <IGF-1R> antibody in PBS (Sigma) including 1% BSA (fraction V, Roche) was added to the wells and incubated for 1-2 h on a microtiterplate shaker at room temperature. For the dilution series the same amount of purified antibody were applied to the wells. The wells were washed three times with 200 |iL/well PBST and bound antibody was detected with 100 |iL/well 30 F(ab')2<hFcy>POD (Dianova) at 0.1 |ig/mL (1:8000) as detection antibody for 1-2 h on a microtiterplate shaker at room temperature. Unbound detection antibody was washed away three times with 200 |iL/well PBST and the bound detection antibody was detected by addition of 100 |iL ABTS/well. Determination of absorbance was performed on a Tecan Fluor Spectrometer at a measurement wavelength of 405 nm 35 (reference wavelength 492 nm). Received at IPONZ on 30.08.2011 - 31 - IGF-IR ECD Biacore Binding of the generated antibodies to human IGF-IR ECD was also investigated by surface plasmon resonance using a BIACORE T100 instrument (GE Healthcare 5 Biosciences AB, Uppsala, Sweden). Briefly, for affinity measurements Goat-Anti-Human IgG, JIR 109-005-098 antibodies were immobilized on a CM5 chip via amine coupling for presentation of the antibodies against human IGF-IR ECD-Fc tagged. Binding was measured in HBS buffer (HBS-P (10 mM HEPES, 150 mM NaCl, 0.005% Tween 20, ph 7.4), 25°C. IGF-IR ECD (R&D Systems or in house 10 purified) was added in various concentrations in solution. Association was measured by an IGF-IR ECD injection of 80 seconds to 3 minutes; dissociation was measured by washing the chip surface with HBS buffer for 3 - 10 minutes and a KD value was estimated using a 1:1 Langmuir binding model. Due to low loading density and capturing level of <IGF-1R> antibodies monovalent IGF-IR ECD 15 binding was obtained. Negative control data (e.g. buffer curves) were subtracted from sample curves for correction of system intrinsic baseline drift and for noise signal reduction. Biacore T100 Evaluation Software version 1.1.1 was used for analysis of sensorgrams and for calculation of affinity data. Figure 11 shows a scheme of the Biacore assay. 20 Examples 1 Production, expression, purification and characterization of monospecific, bivalent <IGF-1R> antibody, wherein the variable domains CL and CHI are replaced by each other (abbreviated herein as <IGF-1R> CL-CH1 exchange antibody). Example 1A 25 Making of the expression plasmids for the monospecific, bivalent <IGF-1R> CL-CH1 exchange antibody The sequences for the heavy and light chain variable domains of the monospecific, bivalent <IGF-1R> CL-CH1 exchange antibody including the respective leader sequences described in this example are derived from a human <IGF-1R> antibody 30 heavy chain (SEQ ID NO: 1, plasmid 4843-pUC-HC-IGF-lR) and a light chain Received at IPONZ on 30.08.2011 - 32- (SEQ ID NO: 2, plasmid 4842-pUC-LC-IGF-lR) described in WO 2005/005635, and the heavy and light chain constant domains are derived from a human antibody (C-kappa and IgGl). The gene segments encoding the <IGF-1R> antibody leader sequence, heavy chain 5 variable domain (VH) and the human kappa-light chain domain (CL) were joined and fused to the 5'-end of the Fc domains of the human yl-heavy chain constant domains (Hinge-CH2-CH3). The DNA coding for the respective fusion protein resulting from the exchange of the CHI domain by the CL domain (CH1-CL exchange) was generated by gene synthesis and is denoted <IGF-1R> HC** (SEQ 10 ID NO: 3) in the following. The gene segments for the <IGF-1R> antibody leader sequence, light chain variable domain (VL) and the human yl-heavy chain constant domain (CHI) were joined as independent chain. The DNA coding for the respective fusion protein resulting from the exchange of the CL domain by the CHI domain (CL-CH1 15 exchange) was generated by gene synthesis and is denoted <IGF-1R> LC** (SEQ ID NO: 4) in the following. Figure 5 and Figure 6 show a schematic view of the protein sequence of the modified <IGF-1R> HC** heavy chain and the modified <IGF-1R> LC** light chain. 20 In the following the respective expression vectors are briefly described: Vector PUC-HC**-IGF-1R Vector pUC-HC**-IGF-IR is an expression plasmid e.g. for transient expression of a CL-CH1 exchange <IGF-1R> heavy chain HC** (cDNA organized expression cassette; with CMV-Intron A) in HEK293 (EBNA) cells or for stable expression in 25 CHO cells. Beside the <IGF-1R> HC** expression cassette this vector contains: - an origin of replication from the vector pUC18 which allows replication of this plasmid in E. coli, and - a 13-lactamase gene which confers ampicillin resistance in E. coli. Received at IPONZ on 30.08.2011 - 33 - The transcription unit of the <IGF-1R> HC** gene is composed of the following elements: - the AscI restriction site at the 5'-end - the immediate early enhancer and promoter from the human cytomegalovirus, 5 - followed by the Intron A sequence, - a 5'-untranslated region of a human antibody gene, - a immunoglobulin light chain signal sequence, - the human <IGF-1R> mature HC** chain encoding a fusion of the human heavy chain variable domain (VH) and the human kappa-light chain constant domain 10 (CL) fused to the 5'-end of the Fc domains of the human yl-heavy chain constant domains (Hinge-CH2-CH3). - a 3' untranslated region with a polyadenylation signal sequence, and - the restriction site SgrAI at the 3'-end. The plasmid map of the heavy chain** CL-CH1 exchange <IGF-1R> HC** 15 expression vector pUC-HC**-IGF-lR is shown in Figure 7. The amino acid sequence of the <IGF-1R> HC** (including signal sequence) is given in SEQ ID NO: 3. Vector PUC-LC**-IGF-1R Vector pUC-LC**-IGF-lR is an expression plasmid e.g. for transient expression of 20 a CL-CH1 exchange <IGF-1R> light chain LC** (cDNA organized expression cassette; with CMV-Intron A) in HEK293 (EBNA) cells or for stable expression in CHO cells. Beside the <IGF-1R> LC** expression cassette this vector contains: - an origin of replication from the vector pUC18 which allows replication of this 25 plasmid in E. coli, and - a 13-lactamase gene which confers ampicillin resistance in E. coli. Received at IPONZ on 30.08.2011 - 34- The transcription unit of the <IGF-1R> LC** gene is composed of the following elements: - the restriction site Sse8387I at the 5' end - the immediate early enhancer and promoter from the human cytomegalovirus, 5 - followed by the Intron A sequence, - a 5'-untranslated region of a human antibody gene, - a immunoglobulin heavy chain signal sequence, - the human <IGF-1R> antibody mature LC** chain encoding a fusion of the human light chain variable domain (YL) and the human yl-heavy chain constant 10 domains (CHI). - a 3' untranslated region with a polyadenylation signal sequence, and - the restriction sites Sail and Fsel at the 3'-end. The plasmid map of the light chain** CL-CH1 exchange <IGF-1R> LC** expression vector pUC-LC**-IGF-lR is shown in Figure 8. The amino acid 15 sequence of the <IGF-1R> LC** (including signal sequence) is given in SEQ ID NO: 4. Plasmids pUC-HC**-IGF-lR and pUC-LC**-IGF-IR can be used for transient or stable co-transfections e.g. into HEK293, HEK293 EBNA or CHO cells (2-vector system). For comparative reasons the wildtype <IGF-1R> antibody was transiently 20 expressed from plasmids 4842-pUC-LC-IGF-lR (SEQ ID NO: 2) and 4843-pUC-HC-IGF-1R (SEQ ID NO: 1) analogous to the ones described in this example. In order to achieve higher expression levels in transient expressions in HEK293 EBNA cells the <IGF-1R> HC** expression cassette can be sub-cloned via AscI, SgrAI sites and the <IGF-1R> LC** expression cassette can be sub-cloned via 25 Sse8387I and Fsel sites into the 4700 pUC-Hyg_OriP expression vector containing - an OriP element, and - a hygromycine resistance gene as a selectable marker. Received at IPONZ on 30.08.2011 - 35 - Heavy and light chain transcription units can either be sub-cloned into two independent 4700-pUC-Hyg-()riP vectors for co-transfection (2-vector system) or they can be cloned into one common 4700-pUC-Hyg-C)riP vector (1-vector system) for subsequent transient or stable transfections with the resulting vectors. Figure 9 5 shows a plasmid map of the basic vector 4700-pUC-OriP. Example IB Making of the monospecific, bivalent <IGF-1R> CL-CH1 exchange antibody expression plasmids 10 The <IGF-1R> fusion genes (HC** and LC** fusion genes) comprising the exchanged Fab sequences of the wildtype <IGF-1R> antibody were assembled with known recombinant methods and techniques by connection of the according nucleic acid segments. The nucleic acid sequences encoding the IGF-IR HC** and LC** were each 15 synthesized by chemical synthesis and subsequently cloned into a pPCRScript (Stratagene) based pGA4 cloning vector at Geneart (Regensburg, Germany). The expression cassette encoding the IGF-IR HC** was ligated into the respective E. coli plasmid via PvuII and BmgBI restriction sites resulting in the final vector pUC-HC**-IGF-lR; the expression cassette encoding the respective IGF-IR LC** 20 was ligated into the respective E. coli plasmid via PvuII and Sail restriction sites resulting in the final vector pUC-LC**-IGF-lR. The subcloned nucleic acid sequences were verified by DNA sequencing. For transient and stable transfections larger quantities of the plasmids were prepared by plasmid preparation from transformed E. coli cultures (Nucleobond AX, Macherey-Nagel) 25 Example 1C Transient expression of monospecific, bivalent IGF-1R> CL-CH1 exchange antibody, purification and confirmation of identity by mass spectrometry Recombinant <IGF-1R> CL-CH1 exchange antibody was expressed by transient co-transfection of plasmids pUC-HC**-IGF-lR and pUC-LC**-IGF-lR in 30 HEK293-F suspension cells as described above. Received at IPONZ on 30.08.2011 - 36- The expressed and secreted monospecific, bivalent <IGF-1R> CL-CH1 exchange antibody was purified from filtered cell culture supernatants by Protein A affinity chromatography according as described above. In brief, the <IGF-1R> CL-CH1 exchange antibody containing cell culture supernatants from transient transfections 5 were clarified by centrifugation and filtration and applied to a Protein A HiTrap MabSelect Xtra column (GE Healthcare) equilibrated with PBS buffer (10 mM Na2HP04, 1 mM KH2P04, 137 mM NaCl and 2.7 mM KC1, pH 7.4). Unbound proteins were washed out with PBS equilibration buffer followed by 0.1 M sodium citrate buffer, pH 5.5 and washed with PBS. Elution of antibody was achieved 10 with 100 mM sodium citrate, pH 2,8 followed by immediate neutralization of the sample with 300(j.l 2 M Tris pH 9.0 per 2 ml fraction. Aggregated protein was separated from monomeric antibodies by size exclusion chromatography on a HiLoad 26/60 Superdex 200 prep grade column (GE Healthcare) in 20 mM Histidine, 150 mM NaCl pH 6.0 and monomeric antibody fractions were 15 subsequently concentrated using a MILLIPORE Amicon Ultra-15 centrifugal concentrator. <IGF-1R> CL-CH1 exchange antibody was frozen and stored at -20°C or -80°C. The integrity of the <IGF-1R> CL-CH1 exchange antibody was analyzed by SDS-PAGE in the presence and absence of a reducing agent and subsequent staining with Coomassie brilliant blue as described above. Monomeric 20 state of the <IGF-1R> CL-CH1 exchange antibody was confirmed by analytical size exclusion chromatography. (Figure 12) Characterized samples were provided for subsequent protein analytics and functional characterization. ESI mass spectrometry confirmed the theoretical molecular mass of the completely deglycosylated <IGF-1R> CL-CH1 exchange antibody. 25 Example ID Analysis of the IGF-IR binding properties of monospecific, bivalent IGF-1R> CL-CH1 exchange antibody in an IGF-IR ECD binding ELISA and by Biacore The binding properties of monospecific, bivalent <IGF-1R> CL-CH1 exchange antibody were evaluated in an ELISA assay with the IGF-IR extracellular domain 30 (ECD) as descried above. For this sake the extracellular domain of IGF-IR (residues 1-462) comprising the natural leader sequence and the Ll-cysteine rich-12 domains of the human IGF-IR ectodomain of the alpha chain (according to the McKern et al., 1997; Ward et al., 2001) fused to an N-terminal His-Streptavidin binding peptide-tag (His-SBP) was cloned into a pcDNA3 vector derivative and 35 transiently expressed in HEK293F cells. The protein sequence of the IGF-IR-His- Received at IPONZ on 30.08.2011 - 37- SBP ECD is given in see above. The obtained titration curve showed that <IGF-1R> CL-CH1 exchange antibody was functional and showed comparable binding characteristics and kinetics as the wildtype <IGF-1R> antibody within the error of the method and thus appeared fully functional (Figure 13). 5 These findings were corroborated by Biacore data with the respective purified antibodies that showed that the monospecific, bivalent <IGF-1R> CL-CH1 exchange antibody with a KD value of 3.7 pM had a comparable affinity and binding kinetics for IGF-IR ECD as the original wildtype <IGF-1R> antibody with a KD value of 3.2 nM: 10 Example 1G Analysis of the IGF-IR binding properties of monospecific, bivalent IGF-1R> CL-CH1 exchange antibody by FACS with IGF-IR over-expressing 124 cells In order to confirm the binding activity of <IGF-1R> CL-CH1 exchange antibody 15 to the IGF-IR over-expressed on the surface of 124 cells (NIH3T3 cells expressing recombinant human IGF-IR, Roche) is studied by FACS. Briefly, 5xl0E5 I24cells per FACS tube are incubated with a dilution of purified <IGF-1R> CL-CH1 exchange antibody and wildtype <IGF-1R> antibody as a reference and incubated on ice for 1 h. Unbound antibody is washed away with 4 ml ice cold PBS (Gibco) + 20 2% FCS (Gibco). Subsequently, cells are centrifuged (5 min at 400 g) and bound antibody is detected with F(ab')2 <hFcy>PE conjugate (Dianova) on ice for 1 h protected from light. Unbound detection antibody is washed away with 4 ml ice cold PBS + 2% FCS. Subsequently, cells are centrifuged (5 min 400 g), resuspended in 300-500 |jL PBS and bound detection antibody is quantified on a 25 FACSCalibur or FACS Canto (BD (FL2 channel, 10.000 cells per acquisition). During the experiment the respective isotype controls are included to exclude any unspecific binding events. Binding of <IGF-1R> CL-CH1 exchange antibody and wildtype <IGF-1R> reference antibody to IGF-IR on 124 cells result in a comparable, concentration dependent shift of mean fluorescence intensity. Received at IPONZ on 30.08.2011 - 38 - Examples 2: Description of a monospecific, bivalent <ANGPT2> wildtype antibody Example 2A Making of the expression plasmids for the monospecific, bivalent <ANGPT2> 5 wildtype antibody The sequences for the heavy and light chain variable domains of a monospecific, bivalent ANGPT2 <ANGPT2> wildtype antibody including the respective leader sequences described in this example are derived from a human <ANGPT2> antibody heavy chain (SEQ ID NO: 6) and a light chain (SEQ ID NO: 7) described 10 in WO 2006/045049 and the heavy and light chain constant domains are derived from a human antibody (C-kappa and IgGl). The wildtype <ANGPT2> antibody was cloned into plasmids SB04-pUC-HC-ANGPT2 (SEQ ID NO: 6) and SB06-pUC-LC-ANGPT2 (SEQ ID NO: 7) that are analogous to the vectors described in the previous example 1 A. 15 For comparative reasons and for co-expression experiments (see example 3) the wildtype <ANGPT2> antibody was transiently (co-)expressed from plasmids SB04-pUC-HC-ANGPT2 and SB06-pUC-LC-ANGPT2. Example 2B Making of the monospecific, bivalent <ANGPT2> wildtype antibody expression 20 plasmids The nucleic acid sequences encoding the ANGPT2> HC and LC were each synthesized by chemical synthesis and subsequently cloned into a pPCRScript (Stratagene) based pGA4 cloning vector at Geneart (Regensburg, Germany). The expression cassette encoding the <ANGPT2> HC was cloned into the respective E. 25 coli plasmid resulting in the final vector SB04-pUC-HC-ANGPT2; the expression cassette encoding the respective <ANGPT2> LC was cloned into the respective E. coli plasmid resulting in the final vector SB06-pUC-LC-ANGPT2. The subcloned nucleic acid sequences were verified by DNA sequencing. For transient and stable transfections larger quantities of the plasmids were prepared by plasmid 30 preparation from transformed E. coli cultures (Nucleobond AX, Macherey-Nagel). Received at IPONZ on 30.08.2011 - 39- Examples 3 Expression of bispecific, bivalent <ANGPT2-IGF-1R> antibody, wherein in the heavy and light chain specifically binding to IGF-IR. the constant domains CL and CHI are replaced by each other (abbreviated herein as <ANGPT2-IGF-1R> CL-5 CHI exchange antibody) Example 3A Transient co-expression and purification of <IGF-1R> CL-CH1 exchange antibody and <ANGPT2> wildtype antibody in HEK293 EBNA cells to yield bispecific <ANGPT2-IGF-1R> CL-CH1 exchange antibody 10 In order to generate a functional bispecific antibody recognizing IGF-IR via the <IGF-1R> CL-CH1 exchange antibody Fab on one side and <ANGPT2> via the <ANGPT2> wildtype Fab region on the other side the two expression plasmids coding for the <IGF-1R> CL-CH1 exchange antibody (example 1A) were co-expressed with two expression plasmids coding for the <ANGPT2> wildtype 15 antibody, (example 2A). Assuming a statistical association of wildtype heavy chains HC and CL-CH1 exchange heavy chains HC** this results in the generation of bispecific and bivalent <IGF-1R-ANGPT2> CL-CH1 exchange antibody . Under the assumption that both antibodies are equally well expressed and without taking side products into account this should result in a 1:2:1 ratio of the three main 20 products A) <IGF-1R> CL-CH1 exchange antibody, B) bispecific <IGF-1R-ANGPT2> CL-CH1 exchange antibody , and C) <ANGPT2> wildtype antibody. Several side products can be expected. However, due to the exchange of only the CL-CH1 domains the frequency of side products should be reduced compared to the complete Fab crossover. Please note as the <ANGPT2> wildtype antibody 25 showed higher expression transient expression yields than the <IGF-1R> wildtype and <IGF-1R> CL-CH1 exchange antibodies the ratio of <ANGPT2> wildtype antibody plasmids and <IGF-1R> CL-CH1 exchange antibody plasmids was shifted in favour of the expression of <ANGPT2> wildtype antibody. To generate the mix of the main products A) <IGF-1R> CL-CH1 exchange 30 antibody, B) bispecific <ANGPT2-IGF-1R> CL-CH1 exchange antibody , and C) <ANGPT2> wildtype antibody the four plasmids pUC-HC**-IGF-lR and pUC-LC**-IGF-1R and plasmids SB04-pUC-HC-ANGPT2 and SB06-pUC-LC-ANGPT2 were transiently co-transfected in suspension HEK293-F cells as described above The harvested supernatant contained a mix of the main products Received at IPONZ on 30.08.2011 -40- A) <IGF-1R> CL-CH1 exchange antibody, B) bispecific <ANGPT2-IGF-1R> CL-CH1 exchange antibody , and C) <ANGPT2> wildtype antibody and is denoted as "Bispecific CL-CH1 exchange mix". Bispecific CL-CH1 exchange mix containing cell culture supernatants, were harvested by centrifugation and subsequently 5 purified as decribed above. Figure 14 The integrity of the antibody mix was analyzed by SDS-PAGE in the presence and absence of a reducing agent and subsequent staining with Coomassie brilliant blue as described. The SDS-PAGE showed that there were 2 different heavy and light chain presents in the preparation as expected (reduced gel). The monomeric state of 10 the antibody mix was confirmed by analytical size exclusion chromatography and showed that the purified antibody species were in a monomeric state. Characterized samples were provided for subsequent protein analytics and functional characterization. Example 3B 15 Detection of functional bispecific <ANGPT2-IGF-1R> CL-CH1 exchange antibody in a cellular FACS bridging assay on 124 IGF-IR expressing cells In order to confirm the presence of functional bispecific <ANGPT2-IGF-1R> CL-CH1 exchange antibody in the purified bispecific CL-CH1 exchange mix of the main products A) <IGF-1R> CL-CH1 exchange antibody, B) bispecific 20 <ANGPT2-IGF-1R> CL-CH1 exchange antibody , and C) <ANGPT2> wildtype antibody from the transient co-expression described in example 3A, a cellular FACS IGF-1R-ANGPT2 bridging assay on 124 cells (NIH3T3 cells expressing recombinant human IGF-IR, Roche) was performed. The assay principle is depicted in Figure 10. A bispecific <ANGPT2-IGF-1R> CL-CH1 exchange 25 antibody that is present in the purified antibody mix is capable of binding to IGF-IR in 124 cells and to ANGPT2 simultaneously; and thus will bridge its two target antigens with the two opposed Fab regions. Briefly, 5xl0E5 I24cells per FACS tube were incubated with total purified antibody mix and incubated on ice for 1 h (titration 160 |jg/ml mix). The respective 30 purified antibodies wildtype <IGF-1R> and <ANGPT2> were applied to the 124 cells as controls. Unbound antibody was washed away with 4 ml ice cold PBS (Gibco) + 2% FCS (Gibco), cells were centrifuged (5 min at 400 g) and bound bispecific antibody was detected with 50 jj.1 2 |ig/mL human ANGPT2 (R&D Received at IPONZ on 30.08.2011 - 41 - Systems) for 1 h on ice. Subsequently, unbound ANGPT2 was washed away once or twice with 4 ml ice cold PBS (Gibco) + 2% FCS (Gibco), cells were centrifuged (5 min at 400 g) and bound ANGPT2 was detected with 50 jj.1 5 \iglmL <ANGPT2>mIgGl-Biotin antibody (BAM0981, R&D Systems) for 45 min on ice; 5 alternatively, cells were incubated with 50 |il 5 |ig/mL mlgGl-Biotin-Isotype control (R&D Systems). Unbound detection antibody was washed away with 4 ml ice cold PBS (Gibco) + 2% FCS (Gibco), cells were centrifuged (5 min at 400 g) and bound detection antibody was detected with 50 |il 1:400 Streptavidin-PE conjugate (Invitrogen/Zymed) for 45 min on ice protected from light. Unbound 10 Streptavidin-PE conjugate was washed away with 4 ml ice cold PBS + 2% FCS. Subsequently, cells were centrifuged (5 min 400 g), resuspended in 300-500 |jL PBS and bound Streptavidin-PE conjugate was quantified on a FACSCalibur (BD (FL2 channel, 10.000 cells per acquisition). During the experiment the respective isotype controls were included to exclude any unspecific binding events. In 15 addition, purified monospecific, bivalent IgGl antibodies <IGF-1R> and <ANGPT2> were included as controls. The results in Fig. 15 show that the incubation with purified antibody crossover mix (<ANGPT2-IGF-1R> CL-CH1 exchange antibody) from the co-expression of a crossover antibody (<IGF-1R> CL-CH1 exchange antibody) with a wildtype 20 antibody (<ANGPT2> wildtype antibody) resulted in a significant shift in fluorescence indicating the presence of a functional bispecific <ANGPT2-IGF-1R> CL-CH1 exchange antibody that was capable of binding to IGF-IR in 124 cells and to ANGPT2 simultaneously; and thus bridges its two target antigens with the two opposed Fab regions. In contrast to this the respective <IGF-1R> and <Ang-2> 25 control antibodies did not result in shift in fluorescence in the FACS bridging assay Taken together these data show that by co-expressing the respective wildtype and crossover plasmids functional bispecific antibodies can be generated. The yields of correct bispecific antibody can be increased by forcing the correct heterodimerization of wildtypoe and modified crossover heavy chains e.g. using 30 the knobs-into-holes technology as well as disulfide stabilization See examples 4) Received at IPONZ on 30.08.2011 -42- Example 4 Expression of bivalent bispecific <ANGPT2-IGF-1R> CL-CH1 exchange antibody with modified CH3 domains (knobs-into-holes) To further improve the yield of the bispecific <ANGPT2-IGF-1R> CL-CH1 5 exchange antibody the knobs-into-holes technology is applied to the co-expression of < IGF-IR > CL-CH1 exchange and wildtype <ANGPT2> antibodies to obtain a homogenous and functional bispecific antibody preparation. For this purpose, the CH3 domain in the heavy chain* HC* of the <IGF-1R> CL-CH1 exchange antibody is replaced by the CH3 domain (Knobs) of the SEQ ID NO: 8 with a 10 T366W exchange and the CH3 domain in the heavy chain of the wildtype <ANGPT2> antibody is replaced by the CH3 domain (Hole) of the SEQ ID NO: 9 with a T366S, L368A, Y407V exchange or vice versa. In addition, a disulfide can be included to increase the stability and yields as well as additional residues forming ionic bridges and increasing the heterodimerization yields 15 (EP 1870459A1). The transient co-expression, and the purification of the resulting bivalent, bispecific <ANGPT2-IGF-1R> CL-CH1 exchange antibody with modified CH3 domains (knobs-into-holes) is performed as described in Example 3. It should be noted that an optimization of heterodimerization can be achieved e.g. 20 by using different knobs-in-holes technologies such as the introduction of an additional disulfide bridge into the CH3 domain e.g. Y349C into the "knobs chain" and D356C into the "hole chain" and/or combined with the use of residues R409D; K370E (K409D) for knobs residues and D399K; E357K for hole residues described by EP 1870459A1. 25 </div>

Claims

<div class="application article clearfix printTableText" id="claims"> Received at IPONZ on 30.08.2011 - 43 - Patent Claims

A bivalent, bispecific antibody, comprising: a) the light chain and heavy chain of an antibody specifically binding to a first antigen; and b) the light chain and heavy chain of an antibody specifically binding to a second antigen, wherein the constant domains CL and CHI are replaced by each other.
The antibody according to claim 1, characterized in that the CH3 domain of one heavy chain and the CH3 domain of the other heavy chain each meet at an interface which comprises an original interface between the antibody CH3 domains; wherein said interface is altered to promote the formation of the bivalent, bispecific antibody, wherein the alteration is characterized in that: a) the CH3 domain of one heavy chain is altered, so that within the original interface the CH3 domain of one heavy chain that meets the original interface of the CH3 domain of the other heavy chain within the bivalent, bispecific antibody, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the interface of the CH3 domain of one heavy chain which is positionable in a cavity within the interface of the CH3 domain of the other heavy chain and b) the CH3 domain of the other heavy chain is altered, so that within the original interface of the second CH3 domain that meets the original interface of the first CH3 domain within the bivalent, bispecific antibody an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the interface of the second CH3 domain within which a protuberance within the interface of the first CH3 domain is positionable; Received at IPONZ on 30.08.2011 -44-
The antibody according to claim 2, characterized in that said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W).
The antibody according to any one of claims 2 or 3, characterized in that said amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), valine (V).
The antibody according to any one of claims 2 to 4, characterized in that both CH3 domains are further altered by the introduction of cysteine (C) as amino acid in the corresponding positions of each CH3 domain.
The antibody according to claim 1, characterized in that one of the constant heavy chain domains CH3 of both heavy chains is replaced by a constant heavy chain domain CHI; and the other constant heavy chain domain CH3 is replaced by a constant light chain domain CL.
A method for the preparation of an a bivalent, bispecific antibody according to claim 1 comprising the steps of a) transforming a host cell, other than a human cell within a human, with -vectors comprising nucleic acid molecules encoding the light chain and heavy chain of an antibody specifically binding to a first antigen -vectors comprising nucleic acid molecules encoding the light chain and heavy chain of an antibody specifically binding to a second antigen, wherein the constant domains CL and CHI are replaced by each other; b) culturing the host cell under conditions that allow synthesis of said antibody molecule; and c) recovering said antibody molecule from said culture. Received at IPONZ on 30.08.2011 - 45 -
8. A host cell comprising: -vectors comprising nucleic acid molecules encoding the light chain and heavy chain of an antibody specifically binding to a first antigen -vectors comprising nucleic acid molecules encoding the light chain and 5 heavy chain of an antibody specifically binding to a second antigen, wherein the constant domains CL and CHI are replaced by each other, and wherein the host cell is not a human cell within a human.
9. A composition of the bivalent, bispecific antibody according to any one of claims 1 to 6.
10 10. A pharmaceutical composition comprising a bivalent, bispecific antibody according to any one of claims 1 to 6 and at least one pharmaceutically acceptable excipient.
11. A bivalent, bispecific antibody according to claim 1 substantially as herein described with reference to any example thereof. 15
12. A method according to claim 7 for the preparation of a bivalent, bispecific antibody substantially as herein described with reference to any example thereof.
13. A host cell according to claim 8 substantially as herein described with reference to any example thereof. 20 Received at IPONZ on 30.08.2011 -1 - SEQUENCE LISTING <110> F. Hoffmann-La Roche AG <120> Bivalent, bispecific antibodies <130> 24679 EP <150> EP 07024865 <151> 2007-12-21 <160> 10 <170> Patentln version 3.2 <210> 1 <211> 467 <212> PRT <213> Artificial <220> <223> amino acid sequence of wild type <IGF-1R> antibody heavy chain <400> 1 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 15 10 15 Val Gin Cys Gin Val Glu Leu Val Glu Ser Gly Gly Gly Val Val Gin 20 25 30 Pro Gly Arg Ser Gin Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Gly Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala lie lie Trp Phe Asp Gly Ser Ser Thr Tyr Tyr Ala 65 70 75 80 Asp Ser Val Arg Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Phe Cys Ala Arg Glu Leu Gly Arg Arg Tyr Phe Asp Leu Trp Gly 115 120 125 Received at IPONZ on 30.08.2011 -2- Arg Gly Thr Leu Val Ser Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 130 135 140 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 180 185 190 Val Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 195 200 205 Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr lie Cys Asn Val Asn His 210 215 220 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie Received at IPONZ on 30.08.2011 - 3 - 340 345 350 Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys 465 <210> 2 <211> 235 <212> PRT <213> Artificial <220> <223> amino acid sequence of wild type <IGF-1R> antibody light chain <400> 2 Met Glu Ala Pro Ala Gin Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 15 10 15 Asp Thr Thr Gly Glu lie Val Leu Thr Gin Ser Pro Ala Thr Leu Ser 20 25 30 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser 35 40 45 Received at IPONZ on 30.08.2011 -4- Val Ser Ser Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro 50 55 60 Arg Leu Leu lie Tyr Asp Ala Ser Lys Arg Ala Thr Gly lie Pro Ala 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser 85 90 95 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Arg Ser 100 105 110 Lys Trp Pro Pro Trp Thr Phe Gly Gin Gly Thr Lys Val Glu Ser Lys 115 120 125 Arg Thr Val Ala Ala Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu 130 135 140 Gin Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 145 150 155 160 Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin 165 170 175 Ser Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser 180 185 190 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 195 200 205 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser 210 215 220 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 3 <211> 471 <212> PRT <213> Artificial Received at IPONZ on 30.08.2011 - 5 - <220> <223> amino acid sequence of the heavy chain** (HC**) of <IGF-1R> CL-CH1 exchange antibody, wherein the heavy chain domain CHI is replaced by the light chain domain CL <400> 3 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 15 10 15 Val Gin Cys Gin Val Glu Leu Val Glu Ser Gly Gly Gly Val Val Gin 20 25 30 Pro Gly Arg Ser Gin Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Gly Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala lie lie Trp Phe Asp Gly Ser Ser Thr Tyr Tyr Ala 65 70 75 80 Asp Ser Val Arg Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Phe Cys Ala Arg Glu Leu Gly Arg Arg Tyr Phe Asp Leu Trp Gly 115 120 125 Arg Gly Thr Leu Val Ser Val Ser Ser Ala Ser Val Ala Ala Pro Ser 130 135 140 Val Phe lie Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly Thr Ala 145 150 155 160 Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val 165 170 175 Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin Glu Ser 180 185 190 Received at IPONZ on 30.08.2011 - 6 - Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr 195 200 205 Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys 210 215 220 Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn 225 230 235 240 Arg Gly Glu Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr 305 310 315 320 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg 355 360 365 Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys 405 410 415 Received at IPONZ on 30.08.2011 - 7 - Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 4 <211> 233 <212> PRT <213> Artificial <220> <223> amino acid sequence of the light chain** (LC**) of <IGF-1R> CL-CH1 exchange antibody, wherein the light chain domain CL is replaced by the heavy chain domain CHI <400> 4 Met Glu Ala Pro Ala Gin Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 15 10 15 Asp Thr Thr Gly Glu lie Val Leu Thr Gin Ser Pro Ala Thr Leu Ser 20 25 30 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser 35 40 45 Val Ser Ser Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro 50 55 60 Arg Leu Leu lie Tyr Asp Ala Ser Lys Arg Ala Thr Gly lie Pro Ala 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser 85 90 95 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Arg Ser 100 105 110 Received at IPONZ on 30.08.2011 Lys Trp Pro Pro Trp Thr Phe Gly Gin Gly Thr Lys Val Glu Ser Lys 115 120 125 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 130 135 140 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 145 150 155 160 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 165 170 175 Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu 180 185 190 Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 195 200 205 Gin Thr Tyr lie Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 210 215 220 Asp Lys Lys Val Glu Pro Lys Ser Cys 225 230 <210> 5 <211> 557 <212> PRT <213> Artificial <220> <223> amino acid sequence of IGF-IR ectodomain His-Streptavidin binding peptide-tag (IGF-IR-His-SBP ECD) <400> 5 Met Lys Ser Gly Ser Gly Gly Gly Ser Pro Thr Ser Leu Trp Gly Leu 15 10 15 Leu Phe Leu Ser Ala Ala Leu Ser Leu Trp Pro Thr Ser Gly Glu lie 20 25 30 Cys Gly Pro Gly lie Asp lie Arg Asn Asp Tyr Gin Gin Leu Lys Arg 35 40 45 Received at IPONZ on 30.08.2011 - 9 - Leu Glu Asn Cys Thr Val lie Glu Gly Tyr Leu His lie Leu Leu lie 50 55 60 Ser Lys Ala Glu Asp Tyr Arg Ser Tyr Arg Phe Pro Lys Leu Thr Val 65 70 75 80 lie Thr Glu Tyr Leu Leu Leu Phe Arg Val Ala Gly Leu Glu Ser Leu 85 90 95 Gly Asp Leu Phe Pro Asn Leu Thr Val lie Arg Gly Trp Lys Leu Phe 100 105 110 Tyr Asn Tyr Ala Leu Val lie Phe Glu Met Thr Asn Leu Lys Asp lie 115 120 125 Gly Leu Tyr Asn Leu Arg Asn lie Thr Arg Gly Ala lie Arg lie Glu 130 135 140 Lys Asn Ala Asp Leu Cys Tyr Leu Ser Thr Val Asp Trp Ser Leu lie 145 150 155 160 Leu Asp Ala Val Ser Asn Asn Tyr lie Val Gly Asn Lys Pro Pro Lys 165 170 175 Glu Cys Gly Asp Leu Cys Pro Gly Thr Met Glu Glu Lys Pro Met Cys 180 185 190 Glu Lys Thr Thr lie Asn Asn Glu Tyr Asn Tyr Arg Cys Trp Thr Thr 195 200 205 Asn Arg Cys Gin Lys Met Cys Pro Ser Thr Cys Gly Lys Arg Ala Cys 210 215 220 Thr Glu Asn Asn Glu Cys Cys His Pro Glu Cys Leu Gly Ser Cys Ser 225 230 235 240 Ala Pro Asp Asn Asp Thr Ala Cys Val Ala Cys Arg His Tyr Tyr Tyr 245 250 255 Ala Gly Val Cys Val Pro Ala Cys Pro Pro Asn Thr Tyr Arg Phe Glu Received at IPONZ on 30.08.2011 -10- 260 265 270 Gly Trp Arg Cys Val Asp Arg Asp Phe Cys Ala Asn lie Leu Ser Ala 275 280 285 Glu Ser Ser Asp Ser Glu Gly Phe Val lie His Asp Gly Glu Cys Met 290 295 300 Gin Glu Cys Pro Ser Gly Phe lie Arg Asn Gly Ser Gin Ser Met Tyr 305 310 315 320 Cys lie Pro Cys Glu Gly Pro Cys Pro Lys Val Cys Glu Glu Glu Lys 325 330 335 Lys Thr Lys Thr lie Asp Ser Val Thr Ser Ala Gin Met Leu Gin Gly 340 345 350 Cys Thr lie Phe Lys Gly Asn Leu Leu lie Asn lie Arg Arg Gly Asn 355 360 365 Asn lie Ala Ser Glu Leu Glu Asn Phe Met Gly Leu lie Glu Val Val 370 375 380 Thr Gly Tyr Val Lys lie Arg His Ser His Ala Leu Val Ser Leu Ser 385 390 395 400 Phe Leu Lys Asn Leu Arg Leu lie Leu Gly Glu Glu Gin Leu Glu Gly 405 410 415 Asn Tyr Ser Phe Tyr Val Leu Asp Asn Gin Asn Leu Gin Gin Leu Trp 420 425 430 Asp Trp Asp His Arg Asn Leu Thr lie Lys Ala Gly Lys Met Tyr Phe 435 440 445 Ala Phe Asn Pro Lys Leu Cys Val Ser Glu lie Tyr Arg Met Glu Glu 450 455 460 Val Thr Gly Thr Lys Gly Arg Gin Ser Lys Gly Asp lie Asn Thr Arg 465 470 475 480 Received at IPONZ on 30.08.2011 -11 - Asn Asn Gly Glu Arg Ala Ser Cys Glu Ser Asp Val Ala Ala Ala Leu 485 490 495 Glu Val Leu Phe Gin Gly Pro Gly Thr His His His His His His Ser 500 505 510 Gly Asp Glu Lys Thr Thr Gly Trp Arg Gly Gly His Val Val Glu Gly 515 520 525 Leu Ala Gly Glu Leu Glu Gin Leu Arg Ala Arg Leu Glu His His Pro 530 535 540 Gin Gly Gin Arg Glu Pro Ser Gly Gly Cys Lys Leu Gly 545 550 555 <210> 6 <211> 471 <212> PRT <213> Artificial <220> <223> amino acid sequence of wild type Angiopoietin-2 <ANGPT2> antibody heavy chain <400> 6 Met Glu Leu Gly Leu Ser Trp Val Phe Leu Val Ala lie Leu Glu Gly 15 10 15 Val Gin Cys Glu Val Gin Leu Val Gin Ser Gly Gly Gly Val Val Gin 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Gly Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ser Tyr lie Ser Ser Ser Gly Ser Thr lie Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn 85 90 95 Received at IPONZ on 30.08.2011 - 12 - Ser Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Asp Leu Leu Asp Tyr Asp lie Leu Thr Gly Tyr 115 120 125 Gly Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 145 150 155 160 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 180 185 190 Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser 195 200 205 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr lie Cys 210 215 220 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 225 230 235 240 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 245 250 255 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 260 265 270 Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 275 280 285 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 290 295 300 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr 305 310 315 320 Received at IPONZ on 30.08.2011 - 13 - Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp 325 330 335 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 340 345 350 Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg 355 360 365 Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 370 375 380 Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 385 390 395 400 lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys 405 410 415 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 420 425 430 Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser 435 440 445 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser 450 455 460 Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 7 <211> 219 <212> PRT <213> Artificial <220> <223> amino acid sequence of wild type Angiopoietin-2 <ANGPT2> antibody light chain <400> 7 Asp lie Val Met Thr Gin Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 15 10 15 Received at IPONZ on 30.08.2011 - 14- Glu Pro Ala Ser lie Ser Cys Arg Ser Ser Gin Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gin Lys Pro Gly Gin Ser 35 40 45 Pro Gin Leu Leu lie Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys lie 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gin Gly 85 90 95 Thr His Trp Pro Pro Thr Phe Gly Gin Gly Thr Lys Leu Glu lie Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu 115 120 125 Gin Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin 145 150 155 160 Ser Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 8 <211> 107 Received at IPONZ on 30.08.2011 - 15 - <212> PRT <213> Artificial <220> <223> amino acid sequence of CH3 domain (Knobs) with a T366W exchange for use in the knobs-into-holes technology <400> Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu 15 10 15 Glu Met Thr Lys Asn Gin Val Ser Leu Trp Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 9 <211> 107 <212> PRT <213> Artificial <220> <223> amino acid sequence CH3 domain (Hole) with a T366S, L368A, Y407V exchange for use in the knobs-into-holes technology <400> 9 Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp 15 10 15 Glu Leu Thr Lys Asn Gin Val Ser Leu Ser Cys Ala Val Lys Gly Phe 20 25 30 Received at IPONZ on 30.08.2011 - 16- Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 10 <211> 557 <212> PRT <213> Artificial <220> <223> amino acid sequence of IGF-IR ectodomain His-Streptavidin binding peptide-tag (IGF-IR-His-SBP ECD) <400> 10 Met Lys Ser Gly Ser Gly Gly Gly Ser Pro Thr Ser Leu Trp Gly Leu 15 10 15 Leu Phe Leu Ser Ala Ala Leu Ser Leu Trp Pro Thr Ser Gly Glu lie 20 25 30 Cys Gly Pro Gly lie Asp lie Arg Asn Asp Tyr Gin Gin Leu Lys Arg 35 40 45 Leu Glu Asn Cys Thr Val lie Glu Gly Tyr Leu His lie Leu Leu lie 50 55 60 Ser Lys Ala Glu Asp Tyr Arg Ser Tyr Arg Phe Pro Lys Leu Thr Val 65 70 75 80 lie Thr Glu Tyr Leu Leu Leu Phe Arg Val Ala Gly Leu Glu Ser Leu 85 90 95 Received at IPONZ on 30.08.2011 - 17 - Gly Asp Leu Phe Pro Asn Leu Thr Val lie Arg Gly Trp Lys Leu Phe 100 105 110 Tyr Asn Tyr Ala Leu Val lie Phe Glu Met Thr Asn Leu Lys Asp lie 115 120 125 Gly Leu Tyr Asn Leu Arg Asn lie Thr Arg Gly Ala lie Arg lie Glu 130 135 140 Lys Asn Ala Asp Leu Cys Tyr Leu Ser Thr Val Asp Trp Ser Leu lie 145 150 155 160 Leu Asp Ala Val Ser Asn Asn Tyr lie Val Gly Asn Lys Pro Pro Lys 165 170 175 Glu Cys Gly Asp Leu Cys Pro Gly Thr Met Glu Glu Lys Pro Met Cys 180 185 190 Glu Lys Thr Thr lie Asn Asn Glu Tyr Asn Tyr Arg Cys Trp Thr Thr 195 200 205 Asn Arg Cys Gin Lys Met Cys Pro Ser Thr Cys Gly Lys Arg Ala Cys 210 215 220 Thr Glu Asn Asn Glu Cys Cys His Pro Glu Cys Leu Gly Ser Cys Ser 225 230 235 240 Ala Pro Asp Asn Asp Thr Ala Cys Val Ala Cys Arg His Tyr Tyr Tyr 245 250 255 Ala Gly Val Cys Val Pro Ala Cys Pro Pro Asn Thr Tyr Arg Phe Glu 260 265 270 Gly Trp Arg Cys Val Asp Arg Asp Phe Cys Ala Asn lie Leu Ser Ala 275 280 285 Glu Ser Ser Asp Ser Glu Gly Phe Val lie His Asp Gly Glu Cys Met 290 295 300 Gin Glu Cys Pro Ser Gly Phe lie Arg Asn Gly Ser Gin Ser Met Tyr 305 310 315 320 Received at IPONZ on 30.08.2011 - 18 - Cys lie Pro Cys Glu Gly Pro Cys Pro Lys Val Cys Glu Glu Glu Lys 325 330 335 Lys Thr Lys Thr lie Asp Ser Val Thr Ser Ala Gin Met Leu Gin Gly 340 345 350 Cys Thr lie Phe Lys Gly Asn Leu Leu lie Asn lie Arg Arg Gly Asn 355 360 365 Asn lie Ala Ser Glu Leu Glu Asn Phe Met Gly Leu lie Glu Val Val 370 375 380 Thr Gly Tyr Val Lys lie Arg His Ser His Ala Leu Val Ser Leu Ser 385 390 395 400 Phe Leu Lys Asn Leu Arg Leu lie Leu Gly Glu Glu Gin Leu Glu Gly 405 410 415 Asn Tyr Ser Phe Tyr Val Leu Asp Asn Gin Asn Leu Gin Gin Leu Trp 420 425 430 Asp Trp Asp His Arg Asn Leu Thr lie Lys Ala Gly Lys Met Tyr Phe 435 440 445 Ala Phe Asn Pro Lys Leu Cys Val Ser Glu lie Tyr Arg Met Glu Glu 450 455 460 Val Thr Gly Thr Lys Gly Arg Gin Ser Lys Gly Asp lie Asn Thr Arg 465 470 475 480 Asn Asn Gly Glu Arg Ala Ser Cys Glu Ser Asp Val Ala Ala Ala Leu 485 490 495 Glu Val Leu Phe Gin Gly Pro Gly Thr His His His His His His Ser 500 505 510 Gly Asp Glu Lys Thr Thr Gly Trp Arg Gly Gly His Val Val Glu Gly 515 520 525 Leu Ala Gly Glu Leu Glu Gin Leu Arg Ala Arg Leu Glu His His Pro Received at IPONZ on 30.08.2011 - 19- 530 535 540 Gin Gly Gin Arg Glu Pro Ser Gly Gly Cys Lys Leu Gly 545 550 555 </div>